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WO1997005286A1 - ANALYSE DE L'EXPRESSION DE GENES PAR MISE EN EVIDENCE DE FRAGMENTS DE RESTRICTION DE L'EXTREMITE 3' D'ADNc - Google Patents

ANALYSE DE L'EXPRESSION DE GENES PAR MISE EN EVIDENCE DE FRAGMENTS DE RESTRICTION DE L'EXTREMITE 3' D'ADNc Download PDF

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WO1997005286A1
WO1997005286A1 PCT/US1996/012468 US9612468W WO9705286A1 WO 1997005286 A1 WO1997005286 A1 WO 1997005286A1 US 9612468 W US9612468 W US 9612468W WO 9705286 A1 WO9705286 A1 WO 9705286A1
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cdna
seq
adapter
display
pcr
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Sherman Weissman
Yatindra Prashar
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Yale University
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Yale University
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Priority to DE69604775T priority Critical patent/DE69604775T2/de
Priority to JP9507853A priority patent/JPH10509329A/ja
Priority to CA002201468A priority patent/CA2201468C/fr
Priority to IL12058296A priority patent/IL120582A0/xx
Priority to AU67636/96A priority patent/AU692403B2/en
Priority to EP96928025A priority patent/EP0797685B1/fr
Publication of WO1997005286A1 publication Critical patent/WO1997005286A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • pairs of 10-mer primers were selected so that each would amplify DNA from about 50 to 100 mRNAs because this number was optimal under this technique for display on the gel [Science 257:967 (1992); and see also United States Patent 5,262,311 , the disclosure of which is incorporated in toto herein].
  • Display patterns are generated when restriction enzyme digested double stranded (ds) cDNA is ligated to an adapter that mediates selective PCR amplification of 3'-end fragments of cDNAs under high stringency PCR conditions, instead of non-stringent arbitrary cDNA amplification as taught by Liang and Pardee.
  • ds restriction enzyme digested double stranded
  • a diversity of patterns is generated by choosing different sets of restriction enzymes and anchored oligo-dT primers.
  • the aspect of the present invention is to describe an assay method that, when compared to presently used methods for such assays, achieves a decrease in false positive signals; provides a reproducible technique for identifying and displaying gel patterns of DNA; provides a means for distinguishing multiple sequence signals for sequences that co- migrate to a single band in conventional techniques; avoids under representation and redundancy of RNA; and utilizes stringent conditions for PCR annealing.
  • Figure 1 is an outline schematic of the method for 3'-end cDNA amplification according to the present invention
  • Figure 2 is a photomicrograph demonstrating the predictability of the band size of known cDNAs on the display gel according to the present invention
  • Figure 3 is a photomicrograph demonstrating the reproducibility patterns and their diversity generated by different restriction enzymes and anchors in oligo-dT primers with heel according to the present invention
  • Figure 4 is a photomicrograph demonstrating a verification of differences from display gel by RT-PCR of original total RNA sample according to the present invention
  • Figure 5 is a photomicrograph showing utility when using a low amount of cDNA for display according to the present invention.
  • Figure 6 is a photomicrograph showing the display obtained from cDNA expression patterns in a very small number of stem cells according to the present invention.
  • Figure 7 is a photomicrograph showing the changes in gene expression patterns found in aging IMR 90 cells and using restriction enzyme Bglll according to the present invention
  • Figure 8 is a photomicrograph showing the display obtained of cDNA expression patterns from human osteoblasts treated with and without estrogen according to the present invention
  • Figure 9 is a photomicrograph showing the changes in gene expression patterns found in aging IMR 90 cells and using restriction enzyme PStl according to the present invention.
  • Figure 10 is a photomicrograph showing the display of cDNA expression patterns of Jurkat T-cells after transfection with the HOX11 gene according to the present invention.
  • RNA expression during early T-cell activation an extensively studied phenomenon associated with the induction of a large number of genes within a relatively short period of time [see Annu. Rev. Immunol. 8:421 (1990); and Curr. Opinion Immunol. 7:327 (1995)] was studied as the test system.
  • Curiously there is a limited description of genes that are down-regulated upon T-cell activation [see Nucleic Acids Res. 19:4655 (1991); and Proc. Natl. Acad. Sci. USA 90:10260 (1993)] and the present approach offered a convenient method for looking for such products.
  • a scheme for the 3'-end restriction fragment display of cDNAs according to the present invention may be described by a general 5-step or more specific 7-step process.
  • step 1 involves the use of a two base anchored oligo-dT primer with a heal for first strand cDNA synthesis from total RNA using reverse transcriptase.
  • all reagents of the reaction except reverse transcriptase are mixed, overlayered with mineral oil and heated at 65° C for 7 minutes to allow opening of any secondary structures formed in the mRNA. Thereafter, the temperature is lowered to 50° C for 7 minutes to allow the anchored oligo-dT primer to anneal to the mRNAs.
  • the reaction mixture from the previous step is used as the substrate for the second strand cDNA synthesis by the Gubler- Hoffman method at 16° C for 2 hours. All cDNA molecules synthesized thus acquire a common 3'-heel as shown in Figure 1. Synthesized double stranded cDNAs are recovered by phenol-chloroform extraction of the reaction mixture, precipitated by ammonium acetate and ethyl alcohol, and dissolved in water.
  • cDNAs from step 2 are digested with a restriction enzyme.
  • the restriction enzyme digested cDNAs are ligated to a ⁇ "-shaped adapter as shown in Figure 1.
  • This adapter has an overhang on its 3'-end for ligation, and on the 5'-end it has a stretch of non-complementary sequence on the opposite strands giving rise to its "Y" shape.
  • the 3'-end restriction fragments will have adapter only on their 5'-end, while internal restriction fragments of the cDNAs will have the adapter ligated to their 5'- and 3'-ends.
  • the 3'-end restriction fragments of the cDNAs ligated to the Y-shaped adapter are selectively amplified.
  • the 5' PCR primer used is made from the Y region of the adapter which does not have the complementary sequence on the opposite strand and, therefore, cannot anneal to the adapter itself.
  • the upstream fragments of digested cDNA with adapter ligated on both ends or only one end in the 5'-terminal piece will, therefore, not be PCR-amplified.
  • the 3' PCR primer (made from the 3'-end heel sequence) anneals to the heel of the 3'-end fragments of cDNA during the first PCR cycle, and extends DNA synthesis into the Y region of the ligated adapter, thus synthesizing complementary sequences to which the 5' PCR primer can now anneal.
  • the two PCR primers can then selectively amplify the 3'-end fragments of the cDNA under stringent conditions.
  • FIG. 1 A two base anchored, oligo-dT primer with a 3' heel is used for first strand synthesis by Gubler-Hoffman method [see Gene 25:263 (1983)] All cDNA molecules thus acquire a common 3' heel.
  • This cDNA is digested with a restriction enzyme and ligated to a Y-shaped adapter similar in principle to the bubble adapter [see Nucleic Acids Res. 18:2887 (1990)].
  • the "Y" adapter is synthesized with an overhang on its 3'-end for ligation, and on its 5'-end it has a stretch of non-complementary sequence on the opposite strands that provides for its 'Y' shape.
  • the 5' PCR primer is synthesized from this Y region and cannot anneal to the adapter itself.
  • the upstream fragments of digested cDNA with adapter ligated on both the ends, or only one end in the 5' terminal piece will, therefore, not be PCR amplified.
  • the 3' primer anneals to the heel of the 3'-end fragments of cDNA during the first PCR cycle and extends DNA synthesis into the Y region of the ligated adapter, thus synthesizing complementary sequences to which the 5' PCR primer can now anneal.
  • the two PCR primers can then selectively amplify the 3'-end fragments of the cDNA under stringent PCR conditions. More specifically there is shown in Figure 1 a method whereby a two base anchored oligo-dT primer with an added heel is used for first strand cDNA synthesis from total RNA using reverse transcriptase (step 1). This is followed by a second strand synthesis using the Gubler-Hoffman method (step 2).
  • cDNA molecules synthesized according to these two steps will thus acquire a common 3' heel.
  • the cDNA is digested (step 3) with a selected restriction enzyme (at this point restriction enzymes that produce either blunt ends or overlapping ends may be used, however, in the following examples the enzyme BstYI is depicted which produced overhanging ends) and ligated to a previously synthesized Y-shaped adapter (step 4).
  • the adapter has an overhang on its 3'-end for ligation, and on its 5'-end it carries a stretch of non-complementary sequences that provide for its shape.
  • the 5' PCR primer is made from this Y region (step 4) and thus cannot anneal to the adapter itself.
  • the upstream fragments of digested cDNA with adapter ligated on both the ends (step 5) or only one end in the 5' terminal piece will, therefore, not be PCR amplified.
  • the 3' primer will anneal to the heel of the 3'-end fragments of cDNA (step 5) during the first PCR cycle and extend DNA synthesis into the Y region of the ligated adapter, thereby synthesizing complementary sequences to which the 5' PCR primer can now anneal (step 6).
  • the two PCR primers can then selectively amplify the 3'-end fragments of the cDNA under stringent conditions (step 7).
  • oligo-dU primers may be used in lieu of the oligo-dT primers for first strand cDNA synthesis in step 1.
  • the binding of oligo-dU to the polyA stretch is known to be weaker when compared to the binding by oligo-dT.
  • the use of oligo-dU primers for first strand cDNA synthesis should minimize the annealing of the oligo-dU to any shorter internal polyA stretches present in the mRNA polymer.
  • the preliminary results from a set of experiments where oligo-dU was used showed typical display patterns with a known sample of RNA.
  • each six base cutter restriction enzyme cuts approximately 8% of the cDNAs at positions between 50 and 400 bases from the polyA tract, so that more than 12 six base cutters will be needed to approach complete representation of cDNAs, each being used with several different anchored oligo-dT primers.
  • cDNA from resting and hr activated human peripheral blood T-lymphocyte RNA were made using oligo- dT primer with a heel and 3' anchor residues TA complementary to the AT dinucleotide in the IL-2 mRNA sequence immediately preceding the polyA tail. Restriction digestion with BstYI should produce, and did when tested, a 146 bp 3'-end fragment of IL-2 cDNA [see Natl. Acad. Sci. USA 81 :2541
  • EXAMPLE 1 Conditions for growth and activation of Jurkat (Jurkat T-cells grow rapidly in culture, can be synchronized by serum starvation, and can be stimulated to produce interleukin-2, an early event in the differentiation of peripheral blood T-cells in vivo; as a result they have become a recognized model system for the comprehensive identification and cloning of all DNA sites that are specifically recognized by protein factors present in these cells; the use of Jurkat T-cells as a model is well accepted, and the findings using this test system may readily be extrapolated to other test systems using other cell types; in short, it is scientifically reasonable to accept Jurkat T-cells as a standard for the comprehensive identification and cloning of all DNA sites that are specifically recognized by protein factors present in these cells, and this may be extrapolated for the comprehensive identification and cloning of all DNA sites that are specifically recognized by protein factors present in other cell types) and peripheral blood T-cells, respectively, have been previously described [see Proc.
  • RNAzol RNAzol
  • the reaction mixture for first strand synthesis included, 10 ⁇ g total RNA, 2 pmols of one of the following three nucleotide anchored heeled oligo-dT primers (wherein T18 refers to a string of 18 thymine-based nucleotides): RP5.0 CTCTCAAGGA TCTTACCGCT Ti sAT 40 (SEQ. NO. 2);
  • the "true” heel is to be considered to be the first 20 nucleotides (that is the molecule without the T ⁇ AT, Ti ⁇ CG, or Ti ⁇ GA portion.
  • the "true” heels are CTCTCAAGGATCTTACCGCT 20 (SEQ. NO. 10); TAATACCGCG CCACATAGCA 20 (SEQ. NO. 11 ); and CAGGGTAGAC GACGCTACGC 20 (SEQ. NO. 12).
  • the reaction mixture was layered with mineral oil, incubated at 65° C for 7 minutes followed by 50° C for another 7 minutes. At this stage 2 ⁇ l of Superscript reverse trascriptase (200 u/ ⁇ l, Gibco-BRL) was added quickly, mixed, and the reaction continued for an additional hour at 50° C. Second strand synthesis was performed at 16° C for 2 hours. At the end of the reaction the cDNAs were precipitated with ethanol and the yield (approximately 100 ng) of cDNA was calculated.
  • oligonucleotide A1 was first kinased at the 5'-end in a final reaction volume of 10 ⁇ l using T4 polynucleotide kinase (PNK) with conventional techniques. After phosphorylation, PNK was heat denatured, and 1 ⁇ g of the oligo A2 was added along with 10X annealing buffer (1M NaCl, 100 mM Tris, HCl pH 8.0, and 10 mM EDTA pH 8.0) in a final working volume of 20 ⁇ l. This mixture was then heated at 65° C for 10 minutes, followed by slow cooling to room temperature for 30 minutes.
  • PNK polynucleotide kinase
  • the ligation was carried out for 16 hrs at 15° C in a final volume of 5 ⁇ l (2 ⁇ l digested cDNA, 1 ⁇ l adapter, and 2 ⁇ l of a solution containing 5 ⁇ l 10 mM ATP, 5 ⁇ l 10X ligation buffer, and 10 ⁇ l (4 units) of T4 DNA ligase). Following ligation the reaction mixture was diluted to a final volume of 80 ⁇ l (adapter ligated cDNA cone. ⁇ 50 pg/ ⁇ l) and heated at 65° C for 10 minutes to denature T4 DNA ligase, and 2 ⁇ l aliquots (with ⁇ 100 pg cDNA) were used for PCR.
  • oligo A1.1 was 5'-end labeled for 30 minutes in a final reaction volume of 20 ⁇ l containing 24 pmoles of this oligo, 10 units of PNK, and 48 pmoles, i.e., 15 ⁇ l of 732p ATP (Amersham).
  • PNK was heat denatured at 65° C for 20 minutes and the labelled oligo was mixed with 60 ⁇ l of 2 ⁇ M of unlabelled oligo 1.1 (at a 1 :4 dilution, final oligo concentration of ⁇ 2 ⁇ M).
  • the PCR reaction mixture (20 ⁇ l) consisted of 2 ⁇ l ( ⁇ 100 pg) of the template, 2 ⁇ l of 10X PCR buffer (100 mM Tris-HCI and 500 mM KCI), 2 ⁇ l of 15 mM MgCl2 to yield 1.5 mM final Mg +2 concentration that is optimum for the reaction, 200 ⁇ M of dNTPs, 200 nM each of 5' and 3' PCR primers, and 1 unit of Amplitaq. Primers and dNTPs were added after preheating the reaction mixture containing the remainder of the components at 85° C.
  • This "hot start” PCR was done to avoid artefactual amplification arising out of arbitrary annealing of PCR primers at Iower temperature during transition from room temperature to 94° C in the first PCR cycle.
  • PCR consisted of 28 to 30 cycles at the stringent conditions of 94° C for 30 seconds, 56° C for 2 minutes, and 72° C for 30 seconds. A higher number of cycles resulted in smeared gel patterns.
  • the PCR amplification conditions may be modified in order to achieve an even higher stringency for primer annealing by using stretch-PCR conditions.
  • stretch-PCR conditions we have routinely used radio ⁇ labelled primer A1 (i.e., Sequence No. 5) as the 5' PCR primer instead of the primer A1.1 described above.
  • the PCR conditions for this stretch-PCR procedure are 94° C for 30 seconds, 68° C for 2 minutes, and 45 seconds for 30 cycles. The results obtained by using this protocol showed improved sharpness of the bands on the display gels.
  • PCR products (2.5 ⁇ l) were analyzed on a 6% polyacrylamide sequencing gel.
  • 13.2 ⁇ l of the ligated cDNA sample was digested with secondary restriction enzymes in a final volume of 20 ⁇ l. From this solution, 3 ⁇ l was used as the template for PCR.
  • This template volume of 3 ⁇ l carried ⁇ 100 pg of the cDNA and 10 mM MGCI2 (from the 10X enzyme buffer) which diluted to the optimum of 1.5 mM in the final PCR volume of 20 ⁇ l. Since any Mg +2 comes from the restriction enzyme buffer, it was not included in the reaction mixture when amplifying secondarily-cut cDNA.
  • bands were extracted from the display gels, reamplified using the 5' and 3' primers, and subcloned into pCRscript with high efficiency using the PCR script cloning kit (Stratagene) and following the manufacturer's instructions. Resulting plasmids were sequenced by cycle sequencing on an Applied Biosystems automated sequencer.
  • 33 P is substituted as the radio-labelling isotope rather than 32p as described above.
  • the isotope 33p emits weak beta particles and has a longer half-life than 32p when the use of 33p W as tested for labelling the PCR primers in lieu of 32p ? he resulting display patterns showed very sharp bands as compared to their 32p counterparts. Based upon these series of comparisons, 33p labelling should allow a better resolution of finer bands that would normally be hidden or not discernible as a result of smudging when 32p j s use d.
  • the display of BstYI digested cDNA prepared from resting and activated T-cells produced the predicted 209 nucleotide band corresponding to IL-2 mRNA 3' end sequence in activated T-cells (lane 2), but not in resting T-cells (lane 1). This is because the IL-2 gene will express the corresponding mRNA in the activated, but not in the resting, T- cells.
  • the total cDNA for the 209 nucleotide sequence is: TAGCGTCCGG CGCAGCGACG GCCAGGATCT TTTATGATTC TTTTTGTAAG 50 CCCTAGGGGC TCTAAAATGG TTTCACTTAT TTATCCCAAA ATATTTATTA 100 TTATGTTGAA TGTTAAATAT AGTATCTATG TAGATTGGTT AGTAAAACTA 150 TTTAATAAAT TTGATAAATA TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT CGCCATTCTA 200 GGAACTCTC 209 (SEQ. NO. 8)
  • nucleotides 1 to 25 correspond to A1
  • nucleotides 26 to 171 correspond to the 146 nucleotide sequence of IL-2 as reported in the literature
  • nucleotides 172 to 209 correspond to the nucleotides of RP5 beginning with the T18 designation.
  • RNA from resting and 4 hour activated Jurkat cells was next compared and displayed in Figure 3 which describes the reproducibility of display patterns and their diversity generated by different restriction enzymes and anchors in oligo-dT primers with heel.
  • Figure 3 depicts three different panels (A, B and C).
  • lanes 1 and 2 are the same experiment, run at different times, using resting cells; lanes 3 and 4 are the same experiment, run at different times, using activated cells.
  • the even numbered lanes are the results of experiments using material from activated T-cells, and the odd numbered lanes are the results of experiments using material from resting T-cells.
  • lanes 1 and 2 are results from an experiment using a first anchor primer; lanes 3 and 4 are results of the same experiment using a different primer.
  • lanes 1 to 4 it is readily apparent that by utilizing the same restriction enzyme, but different anchor primers, one will obtain bands of different proteins.
  • lanes 5 and 6 , and 7 and 8 are results of the same experiments and indicate that with the same cDNA but with different restriction enzymes one will obtain different gel patterns.
  • lanes 9 and 10 are control lanes in which cDNA was cut with restriction enzymes but no adapter or adapter-ligation took place. Thus, lanes 9 and 10 are clear as would be expected of such controls.
  • panel C of Figure 3 it was found to be possible for the first time to resolve sequences which because they share a common weight but not a common sequence migrate together in the gel. Briefly, this was accomplished by randomly introducing a second restriction enzyme into the reaction mixture after ligation and before amplification by PCR. This will not, statistically, cut strands at the same point in both species of cDNA. Thus, statistically the 3'-end obtained in both species will be different in the number of nucleotides.
  • the bands seen on the gel for experiments utilizing different restriction enzymes can, as described above, be used to determine the different genes that are up- or down-regulated.
  • the band identified as JkA5 in panel C is present in all lanes indicating that the gene is active in all preparations tested of active or resting cells.
  • the band identified as JkA6 is present in lane 4 but not lane 3 indicating that this gene is active in activated cells, but is not active in resting cells; thus providing a means of determining the state of the cell.
  • the band identified as JkA7 is the same in lanes 1 , 2 and 4, but is not present in lane 3; again a means is provided for distinguishing between the state of cells based upon the restriction enzyme used.
  • the band identified as JkA7 is present in lanes 1 and 3, the resting cells, but not in the activated cells which provides a means for determining the status of the cell under examination
  • panel A shows that reproducible patterns were observed on display of Bglll digested cDNAs prepared by using oligo-dT primer RP 6.0 from untreated (lanes 1 , 2) and activated (lanes 3, 4) Jurkat RNA samples isolated in two separate experiments.
  • Panel B shows lanes 1, 3, 5, 7 and 9 as representing cDNA samples from untreated, and lanes 2, 4, 6, 8 and 10 from activated, Jurkat cells.
  • Bglll restriction enzyme
  • different cDNAs made from one RNA sample using oligo-dT primers with different anchor bases produce different display patterns (RP9.2 in lanes 1 and 2 and RP 6.0 in lanes 3 and 4).
  • cDNA made from one oligo-dT primer RP6.0 produces different display patterns. Reproducible display patterns were observed between lanes 5 and 6 and 7 and 8 when each cDNA sample of untreated (lanes 5, 7) and activated (lanes 6, 8) Jurkat cells were digested with BamHI in separate tubes and ligated separately before PCR amplification. When aliquots of restriction enzyme digested cDNAs were separately ligated to the adapter at different times identical patterns were observed on PCR amplification. Lanes 9 and 10 represent unligated controls wherein restriction enzyme digested cDNA is utilized for PCR without ligating the adapter.
  • bands may appear in these control lanes if there is a contamination of solutions or samples with adapter ligated cDNA.
  • All samples in panels A and B were run on the same gel, however, lanes 1-4 in panel A, 1 and 2 and 3-10 in panel B were run in adjacent lanes on the gel.
  • lanes 1 and 3 represent untreated, and lanes 2 and 4 represent activated, Jurkat cDNAs prepared by using oligo-dT primer RP6.0.
  • the cDNA was digested with Bglll while in lanes 3 and 4 Bglll digested and adapter ligated cDNA was redigested with a more frequent cutter restriction enzyme, HinF1 , before PCR amplification.
  • JkA6 and JkA7 in lane 4 were revealed as differences on the display gel only upon recutting the Bgl II digested cDNA from lanes 1 and 2 with HinF1 before PCR amplification.
  • recutting Another advantage of recutting is that recovery of low abundance cDNAs is enhanced because removal of high abundance bands by recutting allows access of these fragments to PCR primers ( Figure 3, panel C, lanes 3 and 4).
  • recutting can be used to minimize redundancy between fragments in different lanes. For example, Bglll cut cDNA fragments can be recut with BamHI and vice versa, so that the two samples share no amplified products.
  • a large number of variations of the display patterns can, therefore, be produced by the method according to the present invention to look for differentially expressed genes by (i) a combination of a number of different two base anchored, heeled oligo dT for making cDNAs, (ii) a number of different restriction enzymes that can be used for primary cutting of these cDNAs and, (iii) the number of restriction enzymes used for secondary cutting for each primary cut.
  • RNA and RT-PCR have been described [see J. Exp. Med. 174:1259 (1991)]. Briefly, I ⁇ g of total RNA was reverse transcribed using 100 ng of random hexamer primer in a total volume of 20 ⁇ l.
  • This provides a means for successfully conducting a display study using a smaller-than-presently- required amount of cells from which to obtain the sample material; this application will help to extend the present invention to display of the cDNAs obtainbed for 10 ⁇ g of total RNA to 30 restriction enzymes, in other words, using the present invention 30 different restriction enzymes may be used to successfully display the cDNA prepared from 10 ⁇ g of total RNA.
  • Lanes 1 and 2 in Figure 5 show the display patterns when a normal amount of cDNA is taken (i.e., the amount called for in present display protocols) and subjected to the protocol according to the present invention; lanes 3 and 4 show the display patterns when 1/4th of the amount of cDNA that was used in lanes 1 and 2 was restriction enzyme digested, ligated to the Y- shaped adaptor but diluted 1/4th less than the dilution used in lanes 1 and 2 after ligation in order to obtain the equivalent concentration of the cDNA as when a normal amount of cDNA is taken as in lanes 1 and 2; lanes 5 and 6 show the display patterns of cDNA which was digested as for the samples run in lanes 1 and 2, but only 1/4th of the cDNA was taken for ligation and displayed; and lanes 7 and 8 show the display when 1/4th the amount of ligated cDNA that was used in lanes 1 and 2 was taken for PCR amplification.
  • the results clearly show that display patterns identical to those obtained with amounts
  • FIG. 6 there is shown a display pattern of cDNA obtained from a very small number of sample cells. More specifically, the figure shows comparisons of the cDNA expression pattern in stem cells at different stages of maturation.
  • total RNA was extracted from 5000 stem cells.
  • oligo-dT primer a double stranded cDNA was synthesized, polished, and ligated to an adapter in accordance with the present invention.
  • the adapter primers the cDNA was PCR amplified using the protocol of Baskaran and Weissman [see Genome Research 6(7): 633 (1996)], the disclosure of which is incorporated in toto herein.
  • the original cDNA was therefore amplified several fold so that a large quantity of this cDNA was available for use in the display protocol according to the present invention.
  • an aliquot of this cDNA was incubated with the anchored oligo-dT primer. This mixture was first heat denatured and then allowed to remain at 50° C for 5 minutes to allow the anchor nucleotides of the oligo-dT primers to anneal. This provides for the synthesis of cDNA utilizing a klenow DNA polymerase.
  • the 3'-end region of the parent cDNA (mainly the polyA region) that remains single stranded due to pairing and subsequent synthesis of cDNA by the anchored oligo-dT primer at the beginning of the polyA region, is removed by the 5'-3' exonuclease activity of the T4 DNA polymerase.
  • dNTPs were added in the reaction mixture so that the T4 DNA polymerase would initiate synthesis of the DNA over the anchored oligo-dT primer carrying the heel.
  • the net result of this protocol is that the cDNA with the 3' heel is synthesized for display from the double stranded cDNA as the starting material, rather than RNA as the starting material as occurs in conventional 3'-end cDNA display protocol.
  • the cDNA carrying the 3'-end heel is then subjected to restriction enzyme digestion, ligation, and PCR amplification followed by running the PCR amplified 3'-end restriction fragments with the Y-shaped adapter on the display gel.
  • FIG. 7 there is shown the display changes which occur in the gene expression patterns of the aging human IMR 90 fibroblast cell line.
  • the human fibroblast cell line IMR 90 was grown through passages 7, 13 and 22, after which the cells were harvested and total RNA was isolated as described above. These RNA samples were then used for the display protocol according to the present invention.
  • the cDNA that were synthesized following this protocol were digested with the restriction enzyme Bgl II, and run on display gels that are shown in the figure.
  • the arrows on the display pattern depicted in Figure 7 shows the position of the 3'-end Bgl II fragment of P21 and L7 cDNAs that are known to change during aging.
  • Figure 9 is similar to Figure 7 with the exception that the gel depicted in the figure was run with a cDNA that was digested with the restriction enzyme Pst I. In this case, the arrows show the position of bands in the display pattern that change during aging. Both Figures 7 and 9 thus support the use of the present invention for studies involving the changes which occur in gene expression during the maturation or aging of cells.
  • Figure 8 depicts the gene expression changes that occur in cells exposed to exogenous molecules such as the hormone estrogen.
  • osteoblasts form a 17 year old female patient were obtained from spinal bone chips by allowing cells to grow out of the chips onto plastic culture dishes after which the cells were passaged twice and allowed to grow to confluence in the dishes.
  • cDNA was prepared from control RNA(-) or estrogen treated RNA(+) cells which were digested with Bgl II or Pst I restriction enzymes and run on the display gel as described above. As indicated by the arrow in the figure, this protocol results in a gel that shows the position of one band which is present in the treated cells (i.e., RNA(+)), but absent in the control cells.
  • the protocol of the present invention may also be used to distinguish the effect that an exogenous molecule may have upon various cell types.
  • the display of cDNA expression pattern of Jurkat T-cells after transfection with the HOX11 gene is shown in Figure 10. It is known that the overexpression of the HOX11 gene in T-cells results in leukemia [see Blood (Suppl.) 80:355a (1992)]. In order to study which cDNAs might be induced in T-cells as a result of HOX11 overexpression, the cDNA for HOX11 was transfected into Jurkat cells according to standardized protocol in a tet expression vector [see Proc. Natl. Acad. Sci.
  • lane 1 contained a 1 kb marker
  • 2 contained cDNA samples taken from Jurkat T-cells transfected with the vector plasmid ptet-tak (tak is the plasmid vector that contains the tet-VPIG fusion gene used for transactivation and under tet control; a control)
  • lane 3 contained cDNA samples taken from Jurkat T-cells transfected with the vector plasmid ptet-splice (a control)
  • lane 4 contained cDNA samples taken from Jurkat T-cells transfected with the vector plasmid ptet-HOX11 (a control)
  • lane 6 contained cDNA samples taken from Jurkat T-cells transfected with the vector plasmid ptet-tak +
  • the arrows in Figure 10 indicate some of the cDNA bands that are induced as a result of HOX11 cDNA overexpression, and again support the use of the present invention as a means to locate and identify the initiation of gene expression in cells which have undergone a change from a resting, stable and normal state of existence.
  • Sau3A1 (a four base cutter) produced smeary patterns although the same amount of cDNA digested with Bglll or BamHI (six base cutters) produced clear gel patterns. Sau3a1 is a frequent cutter and produces more amplifiable 3'-ends from a cDNA population which then crowd together on the gel.
  • RNA primed with oligo-dT primers containing a mixture of bases in the subterminal anchor position produced crowded patterns because a larger number of cDNA molecules were synthesized.
  • JkR1 One cDNA , JkR1 , that was down-regulated on activation in both peripheral blood T-cells and Jurkat cells was chosen for further study. Down-regulation of JkR1 in peripheral blood T-cells was evident 4 hours after activation with the addition of ionomycin and diacylglycerol. When resting T-cells were exposed to Actinomycin D, JkR1 mRNA was relatively stable for up to 3 hours. This raises the possibility that part of the down- regulation might be activated by mRNA destabilization.
  • Phillipson has studied a small group of GAS genes whose mRNA declines on refeeding cells with serum [see Cell 54:787 (1988)], and several of these mRNAs were also down-regulated by destabilization [see Mol. Cell Biol. 6:2924 (1990)].
  • JKR1 is different from the GAS genes both in sequence and in the fact that the level of its mRNA was not depressed simply by refeeding but only after specific activation of T-cells.
  • the method of the present invention has shown the ability to uncover multiple differences between rather similar samples of untreated and 4 hr activated Jurkat T-Cells.
  • the reproducibility and extensive representation obtained with this method allows a systematic analysis of a sample, taking full advantage of sequence databases. Since the lane and size of the band of a known gene can be easily predicted, gel patterns can be used to evaluate changes in the level of expression of known mRNAs without resorting to cloning or further analysis. As a large fraction of cDNAs become represented in the databases as 3' ESTs, one can use data base searches to limit or define candidate genes corresponding to any band whose abundance changes. This will be an increasingly powerful approach as more cDNA and genomic sequences accumulate.
  • the present invention is concerned with an approach to study changes in gene expression by selective PCR amplification and display of 3'-end restriction fragments of double stranded cDNAs.
  • the method according to the present invention produces highly consistent and reproducible patterns, can detect almost all mRNAs in a sample, and can resolve hidden differences such as bands that differ in their sequence but comigrate on the gel. Bands corresponding to known cDNAs move to predictable positions on the gel, making this a powerful approach to correlate gel patterns with cDNA databases.
  • Applying the method according to the present invention we have examined differences in gene expression patterns during T-cell activation. Of a total of 700 bands that were evaluated in the T-cell activation studies during the making of the present invention and described herein, as many as 3-4% represented mRNAs that are up-regulated, while about 2% were down-regulated within 4 hours of activation of Jurkat T-cells.
  • oligonucleotide primers are depicted in the embodiments described herein, the exact length and nucleotide sequence of these primers may be varied while still maintaining the requirements that these primers must have as described herein.
  • the embodiment described herein is directed primarily at the assay of changes occurring in activated and resting cells, it is to be understood that whenever a cell is brought into contact with an exogenous material such as for example a pharmaceutical or toxic compound, the cell will invariably provide some genetic response to the material resulting in the up- or down-regulation of genetic expression; these changes can also be noted using the method according to the present invention with very little alteration to the general scheme of the method depicted in Figure 1 , and these changes can be used to evaluate the effect that such materials have on the host cell that has been exposed to the material.
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:1 :
  • MOLECULE TYPE DNA
  • MOLECULE TYPE DNA
  • MOLECULE TYPE DNA
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:10:
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:13:
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:14:

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Abstract

L'invention porte sur une approche consistant à étudier des variations de l'expression de gènes par amplification PCR sélective et mise en évidence des fragments de restriction de l'extrémité 3' d'ADNc double brin.
PCT/US1996/012468 1995-08-01 1996-07-30 ANALYSE DE L'EXPRESSION DE GENES PAR MISE EN EVIDENCE DE FRAGMENTS DE RESTRICTION DE L'EXTREMITE 3' D'ADNc Ceased WO1997005286A1 (fr)

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DE69604775T DE69604775T2 (de) 1995-08-01 1996-07-30 Genexpressionuntersuchung durch aufzeigen von cdna 3'-restriktionsfragmenten
JP9507853A JPH10509329A (ja) 1995-08-01 1996-07-30 ｃＤＮＡの３’末端制限フラグメントの表示による遺伝子発現の分析
CA002201468A CA2201468C (fr) 1995-08-01 1996-07-30 Analyse de l'expression de genes par mise en evidence de fragments de restriction de l'extremite 3' d'adnc
IL12058296A IL120582A0 (en) 1995-08-01 1996-07-30 Analysis of gene expression by display of 3'-end restriction fragments of cDNA
AU67636/96A AU692403B2 (en) 1995-08-01 1996-07-30 Analysis of gene expression by display of 3'-end restriction fragments of cdnas
EP96928025A EP0797685B1 (fr) 1995-08-01 1996-07-30 ANALYSE DE L'EXPRESSION DE GENES PAR MISE EN EVIDENCE DE FRAGMENTS DE RESTRICTION DE L'EXTREMITE 3' D'ADNc

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JPH10509329A (ja) 1998-09-14
AU6763696A (en) 1997-02-26
DE69604775D1 (de) 1999-11-25
EP0797685B1 (fr) 1999-10-20
US6010850A (en) 2000-01-04
EP0797685A1 (fr) 1997-10-01
EP0797685A4 (fr) 1998-06-10
DE69604775T2 (de) 2000-05-18
AU692403B2 (en) 1998-06-04
CA2201468A1 (fr) 1997-02-13
ATE185844T1 (de) 1999-11-15
EP0955379A3 (fr) 2000-03-01
CA2201468C (fr) 2002-03-05
EP0955379A2 (fr) 1999-11-10
IL120582A0 (en) 1997-08-14
US5712126A (en) 1998-01-27

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